Activation of the RAS/RAF/extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway by RAS mutations is commonly found in human cancers. Recently, we reported that mutation of BRAF provides an alternative route for activation of this signaling pathway and can be found in melanomas, colorectal cancers, and ovarian tumors. Here we perform an extensive characterization of BRAF mutations in a large series of colorectal tumors in various stages of neoplastic transformation. BRAF mutations were found in 11 of 215 (5.1%) colorectal adenocarcinomas, 3 of 108 (2.8%) sporadic adenomas, 1 of 63 (1.6%) adenomas from familial adenomatous polyposis (FAP) patients, and 1 of 3 (33%) hyperplastic polyps. KRAS mutations were detected in 34% of carcinomas, 31% of sporadic adenomas, 9% of FAP adenomas, and no hyperplastic polyps. Eight of 16 BRAF mutations were V599E, the previously described hotspot, and none of these was associated with a KRAS mutation in the same lesion. The remaining eight mutations involve other conserved amino acids in the kinase domain, and 62.5% have a KRAS mutation in the same tumor. Our data suggest that BRAF mutations are, to some extent, biologically similar to RAS mutations in colorectal cancer because both occur at approximately the same stage of the adenoma-carcinoma sequence, both are associated with villous morphology, and both are less common in adenomas from FAP cases. By contrast, colorectal adenocarcinomas with BRAF mutations are associated with early Dukes’ tumor stages (P = 0.006) and no such relationship was observed for KRAS mutations. The presence in some colorectal neoplasms of mutations in both BRAF and KRAS suggests that modulation of the RAS-RAF-extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway may occur by mutation of multiple components.

Colorectal cancer develops through a multistep process of mutation and clonal expansion. It has been shown that somatic mutation of the RAS genes, in particular KRAS, is an early event in colorectal carcinogenesis, predominantly occurring during the transformation of a small to intermediate sized adenoma (1).

The RAS proteins participate in the RAS-RAF-MEK-ERK-MAP kinase3 pathway, which mediates cellular responses to growth signals (2). There are three RAF genes, each encoding cytoplasmic serine/threonine kinases that are regulated by binding to RAS (2, 3). We have previously reported that BRAF is somatically mutated in a number of human cancers, including malignant melanoma, colorectal carcinoma, and ovarian borderline (low malignant potential) tumors (4). Mutations in BRAF occur in two regions of the BRAF kinase domain, the G loop (which mediates binding of ATP) and the activation segment (which protects the substrate binding site). Mutated forms of BRAF that have been studied thus far have elevated kinase activity and can transform NIH3T3 cells (4).

We now investigate in greater detail the occurrence and spectrum of BRAF mutations in colorectal cancer and in particular their biological relationship to KRAS mutations.

Colorectal Tumor Samples.

Samples from colorectal cancer surgical resections and polyps were collected at Queen Mary Hospital between 1990 and 2001. All specimens were received fresh from the operating theater or endoscopy room. Representative tumor and normal blocks (if available) were snap frozen in liquid nitrogen and stored at −70°C. Frozen sections were cut for histology evaluation. Blocks with tumor occupying >70% of the section area were used. The remainder of each specimen was fixed in 10% buffered formalin and processed through paraffin for histology. DNA was extracted by standard protocols using proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. The study was approved by the Ethics Committee of the University of Hong Kong.

Mutation Screening.

Screening for mutations was performed as described previously (4). In brief, we used a capillary-based modified heteroduplex method optimized to run on an ABI PRISM 3100 Genetic Analyzer. PCR primers were designed to amplify the exons plus at least 50 bp of flanking intronic sequence (details of the primers used are available in the supplementary information of our previous paper (4). Genomic DNA, 12 ng from the test sample, was mixed with 3 ng of control genomic DNA and amplified using standard PCR conditions in which one of the primers was labeled with FAM, NED, or VIC dye. The resulting samples were then analyzed on an ABI PRISM 3100 Genetic Analyser under semidenaturing conditions using optimized separation medium and run conditions. The resulting traces were analyzed using proprietary software to identify samples that produce a shift in peak migration relative to a standard normal control, indicating the presence of a putative sequence variation. Samples that produced a heteroduplex shift were directly sequenced on both strands using the BigDye terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s protocol and analyzed on an ABI PRISM 3100 Genetic Analyser. Analysis of microsatellites for the presence of instability in colorectal cancers was performed as previously described (5). The following loci were used: BAT26, BAT25, BAT40, TGFβRII (transforming growth factor-β receptor II), D2S123, D5S346, TP53, D18S58, D3S1067, D5S82, and DCC (deleted in colon cancer). At least five loci were analyzed in each case, including both dinucleotide and mononucleotide loci. Tumors were designated high-level microsatellite instability (MSI-H) when at least 40% loci showed altered electrophoretic mobility relative to the corresponding normal tissue. Tumors with some (but <40%) altered loci were designated low-level microsatellite instability (MSI-L), and those without altered loci were designated microsatellite stable (MSS). For colonic polyps, the MSI status was defined by shifts in BAT26 and BAT25 as corresponding normal tissue was not available.

We studied 215 colorectal adenocarcinomas from colectomies (212 cases) or local resections of liver metastases (3 cases); 122 were from male and 93 from female patients. The patients’ age ranged from 24 to 89 years (mean age, 58 years). There were 45 tumors in the right colon and 170 in the left colon. There were 28 Dukes’ A, 71 Dukes’ B, 83 Dukes’ C, and 33 Dukes’ D cancers. Nineteen were well-differentiated, 168 were moderately differentiated, and 28 were poorly differentiated. Twenty of these cases have been included in our previous study reporting the incidence of BRAF mutation in various types of human cancer (4).

We also studied 113 sporadic colorectal polyps; 72 were from male patients, and 41 were from female patients. The patients’ age ranged from 20 to 87 years (mean age, 62 years). There were 66 tubular adenomas, 25 tubulovillous adenomas, 17 villous adenomas, 1 retention polyp, 3 hyperplastic polyps, and 1 Peutz-Jegher polyp. Among these, 4 showed no dysplasia, 32 showed mild dysplasia, 34 showed moderate dysplasia, and 33 showed severe dysplasia. 10 showed early malignant transformation into invasive cancers. 88 were located in the left colon and 24 in the right colon (1 case had unknown tumor location). The size of the polyps ranged from 3 to 45 mm in maximum dimension (mean, 10.88 mm; see Table 6 for size distribution). In addition, 63 polyps from 3 patients with FAP3 were studied. The diagnosis of FAP in these individuals was made on the basis of clinical criteria and confirmed by identification of germline adenomatous polyposis coli mutations (patient 1, deletion exons 4–7; patient 2, c.1349–1355delTCTGTGT; patient 3, c.3284–3285delAG). The FAP polyps ranged from 3 to 20 mm in maximum dimension (mean, 6.3 mm). These tumors were generally small tubular adenomas with low-grade dysplasia (see Table 6 for size distribution); 21 were located in the left colon and 42 were found in the right colon. There were 58 tubular, 1 villous, and 4 tubulovillous adenomas. Forty-two had mild dysplasia, 19 had moderate dysplasia, and 2 had severe dysplasia.

The spectrum of BRAF mutations found in both colorectal adenocarcinomas and colonic polyps, their association with clinicopathological features, and KRAS mutations are shown in Tables 1 and 2. Of 215 colorectal adenocarcinomas studied, there were 11 cases (5.1%) with BRAF mutations (3 of these were previously reported (4). The mutations were absent in the corresponding normal mucosae and hence were somatically acquired. Of these colorectal cancers, 208 were successfully analyzed for KRAS mutations; in 70 (33.7%), a KRAS mutation was identified. The spectrum of KRAS mutations is shown in Table 3.

Of the 11 colorectal cancers with BRAF mutations, 5 were from female and 6 were from male patients. Two arose from the right colon and nine from the left colon. Two were well differentiated, eight were moderately differentiated, and one was poorly differentiated. Two were mucinous adenocarcinomas (from a total of 15 mucinous carcinomas examined). One tumor was Dukes’ stage A (of 27 examined), 9 were Dukes’ stage B (of 71 examined), 1 was Dukes’ stage C (of 83 examined), and none was Dukes’ stage D (of 33 examined) (Table 4). Overall, there was no significant association of BRAF mutations with patient gender, age, tumor differentiation, mucinous morphology, or location. There was a statistically significant association of BRAF mutations with early Dukes’ stages (Dukes’ stage A and B versus C and D, P = 0.006, χ2 test). MSI status was available in 107 cancers. 3 of 11 cancers (27%) with BRAF mutation were MSI-H, compared with 19 of 96 cancers (20%) without BRAF mutations (P = 0.851). We did not observe any association of KRAS mutations with patient gender, age, tumor location, differentiation, mucinous morphology, or tumor stage.

Of the 113 sporadic colonic polyps studied, 4 had BRAF mutations (3.5%). BRAF mutations were found in 3 of 108 adenomas (2.8%), 1 of 3 hyperplastic polyps (33%), and none in the single retention and Peutz-Jegher polyps. We successfully analyzed 108 polyps for KRAS mutations, and in 32 (29.6%) a KRAS mutation was identified [all occurring in the adenomas (31%)]. Of the 63 adenomas from FAP patients, only 1 (1.6%) showed BRAF mutations. We successfully analyzed 54 FAP adenomas for KRAS mutations, and 5 (9%) were positive. The incidence of KRAS mutation was significantly lower in FAP polyps than in sporadic ones (P = 0.004, χ2 test). The incidence of BRAF mutations also appeared lower in FAP adenomas. The spectrum of KRAS mutations in polyps is shown in Table 3.

The relationship of BRAF and KRAS mutations with morphology and size of sporadic and FAP adenomas are summarized in Tables 5 and 6. All three sporadic adenomatous polyps with BRAF mutations showed villous morphology. Although the number of adenomatous polyps with BRAF mutations was small, the association with villous morphology reach borderline statistical significance (P = 0.056, tubular versus villous/tubulovillous, Fisher’s exact test). The trend persisted (P = 0.064) even taking the FAP adenomas into statistical calculation (Table 5). Overall, a modest association for right-sided location was observed; all 4 adenomas (sporadic and FAP) with BRAF mutation arose in the right colon (from a total of 65) and 0 from the left colon (from a total of 105; P = 0.035, Fisher’s exact test). BRAF mutation was absent in sporadic and FAP adenomas <5 mm in diameter (Table 6). There was no significant association of BRAF mutation with patient’s age, gender, polyp size, and degree of dysplasia.

Adenomas with KRAS mutations were more often associated with villous or tubulovillous morphology (Tables 5 and 6; P < 0.001, χ2 test). KRAS mutation was also absent in sporadic and FAP adenomas less than 5 mm in size (Table 6). In addition, adenomas with KRAS mutation were significantly larger (mean size with mutation, 13.51 ± 6.55 mm; mean size without mutation, 8.09 ± 3.85 mm; P < 0.001 by Student t test) and were more common in adenomas with higher grade dysplasia and those with transformation to invasive cancer (8 of 67 mild dysplasia, 11 of 46 moderate dysplasia, 14 of 34 severe dysplasia, and 4 of 10 adenomas with malignant transformation; P = 0.006, χ2 test). Otherwise, there was no association of KRAS with patient age, gender, and location of tumor.

Eight of the 16 BRAF mutations were T1796A (resulting in the substitution of valine 599 by glutamate), a previously documented hotspot (4). None of the colorectal tumors with V599E carried a KRAS mutation. The remaining BRAF mutations were G468E(4), F594L, and six novel mutations, N580S, D593V, D593G, G595R, T598I, and F467C. The novel mutations all changed highly conserved amino acids (Fig. 1). Five of eight tumors containing non-V599E BRAF mutations had KRAS mutations. Although one of these, Q22K, is unusual, it has been previously reported and is associated with transforming activity (6). The incidence of KRAS mutations was significantly higher in colorectal tumors (cancer and polyps) with non-V599E BRAF mutations (five of eight) than in those with V599E mutations (none of 8; P = 0.012, Fisher’s exact test), and also those with no BRAF mutation (102 of 354; P = 0.039, χ2 test).

Several conclusions are suggested by the data presented: (a) BRAF mutations are present predominantly in intermediate sized adenomas. Although the incidence of BRAF mutation in colorectal adenomas (2.7%) was lower than in carcinomas (5.1%), these results were not significantly different and overall suggest that BRAF mutations occur in adenoma stage. The stage in the adenoma-carcinoma sequence at which BRAF mutations arise is therefore similar to that at which KRAS mutations occur; (b) colorectal adenomas with BRAF mutations, similar to those with KRAS mutations, are strongly associated with villous morphology; (c) the frequency of BRAF mutations is lower in adenomas from FAP cases, similar to the pattern for KRAS mutations; (d) colorectal carcinomas with BRAF mutations tend to have a lower Dukes’ stage; and (e) supporting the previously reported observations (4), tumors with BRAF V599E do not have KRAS mutations, whereas other BRAF mutations are often accompanied by KRAS mutations in the same tumors.

We observed a lower incidence of BRAF mutations in colorectal cancer than in our previous study of a few cases (4). The lower incidence is unlikely to be a result of lower sensitivity of the detection methods, given that the same methodology has been used in both studies. Indeed, using this detection method, we found KRAS mutations in 33.7% of cases, an incidence that compared well with our previous series and a compilation of world literature series of KRAS mutations in colorectal cancer (7, 8). Moreover, there were no obvious differences in the stage, grade, or histology of the new cases in this report compared with those previously described (4). Reconciliation of these frequency estimates of BRAF mutations in colorectal cancer will require additional studies.

Our data indicate that BRAF mutations are more common in Dukes’ stage A/B colorectal carcinomas than in Dukes’ stage C/D. This may suggest a less invasive biological behavior for those cancers that harbor BRAF mutations. Follow-up data are available in seven of nine cases with Dukes’ stage B tumors carrying BRAF mutations. They all have survived without recurrence (mean follow-up 60 months, range 5–87) compared with 75% 5-year survival in BRAF-negative Dukes’ B cancers from this series (this comparison does not reach statistical significance). Study of a larger series will be necessary to evaluate further if colorectal cancers with a BRAF mutation are associated with better survival.

We observed many similarities in tumors with BRAF or KRAS mutation that may reflect a common role of the RAS-RAF-extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in colorectal tumorigenesis. BRAF and KRAS mutations occur at the adenoma stage of the adenoma-carcinoma sequence (although there is a statistically nonsignificant tendency in our series for BRAF mutations to be more common in carcinomas than in adenomas). Like KRAS(9), BRAF mutations are preferentially more common in villous and rare in tubular adenomas. Also adenomas arising from FAP patients rarely have KRAS(1) or BRAF mutation. Because the FAP adenomas in our series are generally smaller than the sporadic adenomas and most are tubular adenomas, this may partly explain the rarity of KRAS and BRAF mutations. We also found a BRAF mutation in a hyperplastic polyp and a small percentage of these have previously been shown to harbor KRAS mutations (10, 11). In contrast, the association of BRAF mutations with Dukes’ stage A/B colorectal cancers was not observed for KRAS mutations.

We previously showed that a mutation in the activation segment, conversion of valine 599 to glutamic acid, is a hotspot for BRAF mutation in human cancer (4). This mutation results in the insertion of a negatively charged residue adjacent to a site of regulatory phosphorylation at T598, which may mimic regulatory phosphorylation, thus leading to constitutive activation of BRAF independent of RAS. Here we confirmed that V599E is the most common mutation in colorectal tumors, accounting for 50% of mutation observed. Consistent with the proposed autonomous nature of this mutation in the RAS-RAF-MEK-ERK-MAP kinase signaling pathway (4), mutation of RAS is not required and hence not observed in any of the tumors carrying V599E. We identified six novel BRAF mutations involving other conserved amino acids in the vicinity of the activation segment or glycine-rich loop. Most of these were associated with KRAS mutations in the same cancers. This is consistent with our previous observations on other non-V599 BRAF mutations and suggests modulation of the RAS-RAF-MEK-ERK-MAP kinase signaling pathway by mutation of multiple components in colorectal cancer.

It is interesting to speculate on the functional consequences of three of the novel BRAF mutations, D593V, D593G, and T598I, all located in the kinase activation segment. In BRAF, both T598 and S601 require phosphorylation to achieve maximal kinase activity. Phosphorylation of these two residues occurs after recruitment of BRAF to the membrane by activated RAS. In experimental systems, replacement of these two residues by acidic amino acids can mimic regulatory phosphorylation and results in RAS-independent BRAF activation. However, replacement by nonpolar amino acids results in complete abrogation of BRAF kinase activity (12). Similarly, previously described experimental mutants of BRAF D593 abolish kinase activity. Although all of the cancer-associated BRAF mutants previously studied were associated with increased kinase activity and transforming activity in NIH3T3 cells (4), the current results suggest that some BRAF mutations found in human cancer may reduce or abolish kinase and transforming activities. Clearly, more studies are required to functionally characterize these mutants.

While this paper was being considered for publication, a similar study was published by Rajagopalan et al., Nature, 418: 934, 2002.

Fig. 1.

Sequence conservation and mutations in the BRAF activation domain (A) and G loop (B) in colorectal tumors analyzed in this report (▪) and total of all published cancers with BRAF mutations (□) (4).

Fig. 1.

Sequence conservation and mutations in the BRAF activation domain (A) and G loop (B) in colorectal tumors analyzed in this report (▪) and total of all published cancers with BRAF mutations (□) (4).

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Wellcome Trust, the Institute of Cancer Research, the Research Grants Council of the Hong Kong Special Administrative Region (HKU 7330/00M), the Hong Kong Society of Gastroenterology, and the Hong Kong Cancer Fund.

3

The abbreviations used are: RAS-RAF-ERK-MEK-MAP kinase, RAS-RAF-extracellular signal-regulated kinase-mitogen-activated protein kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase; FAP, familial adenomatous polyposis; MSI, microsatellite instability.

Table 1

Spectrum of BRAF mutation in 11 colorectal cancers, their associated clinicopathological features, and KRAS mutation status

BRAF mutationsSexAge (yr)Tumor sideMSIDukes’ stageMucinousDifferentiationKRAS
Nucleotideaaa
T1796A V(599)E              b 75 MSI-H Yes Moderate Nil 
T1796A V(599)E              b 62 MSS No Moderate Nil 
T1796A V(599)E 67 MSI-H No Poor Nil 
T1796A V(599)E 38 MSS Yes Moderate Nil 
C1793T T(598)I 64 MSI-H No Moderate G13D 
G1783C G595R 62 MSS No Well Nil 
A1778T D593V 43 MSI-L No Moderate G12D 
A1778G D593G 43 MSS No Moderate G13C 
A1739G N580S 87 MSS No Well Q22K 
G1403A G468E              b 76 MSS No Moderate Nil 
T1400G F467C 40 MSS No Moderate G12D 
BRAF mutationsSexAge (yr)Tumor sideMSIDukes’ stageMucinousDifferentiationKRAS
Nucleotideaaa
T1796A V(599)E              b 75 MSI-H Yes Moderate Nil 
T1796A V(599)E              b 62 MSS No Moderate Nil 
T1796A V(599)E 67 MSI-H No Poor Nil 
T1796A V(599)E 38 MSS Yes Moderate Nil 
C1793T T(598)I 64 MSI-H No Moderate G13D 
G1783C G595R 62 MSS No Well Nil 
A1778T D593V 43 MSI-L No Moderate G12D 
A1778G D593G 43 MSS No Moderate G13C 
A1739G N580S 87 MSS No Well Q22K 
G1403A G468E              b 76 MSS No Moderate Nil 
T1400G F467C 40 MSS No Moderate G12D 
a

aa, amino acid.

b

These mutations have been reported in our previous study (4).

Table 2

Spectrum of BRAF mutation in 5 colonic polyps, their associated clinicopathological features, and KRAS mutation status

BRAF mutationsSexAge (yr)Sporadic/FAPTumor sideMSITumor size (mm)MorphologyDysplasiaKRAS
Nucleotideaa
T1796A V(599)E 62 Sporadic MSS 5.5 Hyperplastic Nil Nil 
T1796A V(599)E 49 Sporadic MSS 13 Villous Mild Nil 
T1796A V(599)E 60 Sporadic MSS 10 Villous Moderate Nil 
T1796A V(599)E 65 Sporadic MSS Villous Severe Nil 
T1782G F(594)L 68 FAP MSS Tubular Mild Nil 
BRAF mutationsSexAge (yr)Sporadic/FAPTumor sideMSITumor size (mm)MorphologyDysplasiaKRAS
Nucleotideaa
T1796A V(599)E 62 Sporadic MSS 5.5 Hyperplastic Nil Nil 
T1796A V(599)E 49 Sporadic MSS 13 Villous Mild Nil 
T1796A V(599)E 60 Sporadic MSS 10 Villous Moderate Nil 
T1796A V(599)E 65 Sporadic MSS Villous Severe Nil 
T1782G F(594)L 68 FAP MSS Tubular Mild Nil 
Table 3

Spectrum of KRAS mutation in 70 colorectal cancers, 32 sporadic adenomas, and 5 FAP adenomas

Nucleotide changeAmino acid changeNo. of CRCsNo. of adenomasNo. of FAP adenomas
G35A G12D 21 14 
G35T G12V 15 
G34A G12S   
G34T G12C  
G35C G12A  
G34C G12R   
G38A G13D 19  
G37T G13C  
C64A Q22K   
C176A A59E   
G175A A59T   
A182G Q61R   
A183T, A183C Q61H  
G274T D92Y   
Nucleotide changeAmino acid changeNo. of CRCsNo. of adenomasNo. of FAP adenomas
G35A G12D 21 14 
G35T G12V 15 
G34A G12S   
G34T G12C  
G35C G12A  
G34C G12R   
G38A G13D 19  
G37T G13C  
C64A Q22K   
C176A A59E   
G175A A59T   
A182G Q61R   
A183T, A183C Q61H  
G274T D92Y   
Table 4

Relationship of BRAF mutation with tumor stage in 215 colorectal cancers

Dukes’ stageaBRAF mutation
NegativePositive
27 
62 
82 
33 
Dukes’ stageaBRAF mutation
NegativePositive
27 
62 
82 
33 
a

Stage A and B versus stage C and D, P = 0.006.

Table 5

Relationship of BRAF and KRAS mutation with morphology of sporadic and FAP colorectal adenomas

BRAF mutationKRAS mutation
NegativePositiveNegativePositive
Tubular 123 100 12 
Villous 15 10 
Tubulovillous 29 12 15 
     
P P = 0.064, tubular vs. villous/tubulovillous, Fisher’s exact test  P < 0.001, χ2 test  
BRAF mutationKRAS mutation
NegativePositiveNegativePositive
Tubular 123 100 12 
Villous 15 10 
Tubulovillous 29 12 15 
     
P P = 0.064, tubular vs. villous/tubulovillous, Fisher’s exact test  P < 0.001, χ2 test  
Table 6

Relationship of BRAF and KRAS mutations with size of sporadic and FAP colorectal adenomas

Size of adenoma (mm)Sporadic adenomasAdenomas from FAP patients
BRAF mutation (%)KRAS mutation (%)BRAF mutation (%)KRAS mutation (%)
<5 0 (8)a 0 (8) 0 (17) 0 (15) 
5–10 3 (33) 18.2 (33) 2.4 (42) 5.7 (35) 
10–15 4.3 (47) 40.1 (42) 0 (1) 100 (1) 
>15 0 (20) 45 (20) 0 (3) 66.7 (3) 
     
Total 2.8 (108) 31 (103) 1.6 (63) 9.3 (54) 
Size of adenoma (mm)Sporadic adenomasAdenomas from FAP patients
BRAF mutation (%)KRAS mutation (%)BRAF mutation (%)KRAS mutation (%)
<5 0 (8)a 0 (8) 0 (17) 0 (15) 
5–10 3 (33) 18.2 (33) 2.4 (42) 5.7 (35) 
10–15 4.3 (47) 40.1 (42) 0 (1) 100 (1) 
>15 0 (20) 45 (20) 0 (3) 66.7 (3) 
     
Total 2.8 (108) 31 (103) 1.6 (63) 9.3 (54) 
a

Numbers in parentheses, number analyzed.

1
Vogelstein B., Fearon E. R., Hamilton S. R., Kern S. E., Preisinger A. C., Leppert M., Nakamura Y., White R., Smits A. M., Bos J. L. Genetic alterations during colorectal-tumor development.
N. Engl. J. Med.
,
319
:
525
-532,  
1988
.
2
Peyssonnaux C., Eychene A. The Raf/MEK/ERK pathway: new concepts of activation.
Biol. Cell
,
93
:
53
-62,  
2001
.
3
Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions.
Biochem. J.
,
351 (Pt. 2)
:
289
-305,  
2000
.
4
Davies H., Bignell G. R., Cox C., Stephens P., Edkins S., Clegg S., Teague J., Woffendin H., Garnett M. J., Bottomley W., Davis N., Dicks E., Ewing R., Floyd Y., Gray K., Hall S., Hawes R., Hughes J., Kosmidou V., Menzies A., Mould C., Parker A., Stevens C., Watt S., Hooper S., Wilson R., Jayatilake H., Gusterson B. A., Cooper C., Shipley J., Hargrave D., Pritchard-Jones K., Maitland N., Chenevix-Trench G., Riggins G. J., Bigner D. D., Palmieri G., Cossu A., Flanagan A., Nicholson A., Ho J. W., Leung S. Y., Yuen S. T., Weber B. L., Seigler H. F., Darrow T. L., Paterson H., Marais R., Marshall C. J., Wooster R., Stratton M. R., Futreal P. A. Mutations of the BRAF gene in human cancer.
Nature (Lond.)
,
417
:
949
-954,  
2002
.
5
Chan T. L., Yuen S. T., Chung L. P., Ho J. W., Kwan K. Y., Chan A. S., Ho J. C., Leung S. Y., Wyllie A. H. Frequent microsatellite instability and mismatch repair gene mutations in young Chinese patients with colorectal cancer.
J. Natl. Cancer Inst.
,
91
:
1221
-1226,  
1999
.
6
Tsukuda K., Tanino M., Soga H., Shimizu N., Shimizu K. A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma.
Biochem. Biophys. Res. Commun.
,
278
:
653
-658,  
2000
.
7
Yuen S. T., Chung L. P., Leung S. Y., Luk I. S., Chan S. Y., Ho J. C., Ho J. W., Wyllie A. H. Colorectal carcinoma in Hong Kong: epidemiology and genetic mutations.
Br. J. Cancer
,
76
:
1610
-1616,  
1997
.
8
Andreyev H. J., Norman A. R., Cunningham D., Oates J., Dix B. R., Iacopetta B. J., Young J., Walsh T., Ward R., Hawkins N., Beranek M., Jandik P., Benamouzig R., Jullian E., Laurent-Puig P., Olschwang S., Muller O., Hoffmann I., Rabes H. M., Zietz C., Troungos C., Valavanis C., Yuen S. T., Ho J. W., Croke C. T., O’Donoghue D. P., Giaretti W., Rapallo A., Russo A., Bazan V., Tanaka M., Omura K., Azuma T., Ohkusa T., Fujimori T., Ono Y., Pauly M., Faber C., Glaesener R., de Goeij A. F., Arends J. W., Andersen S. N., Lovig T., Breivik J., Gaudernack G., Clausen O. P., De Angelis P. D., Meling G. I., Rognum T. O., Smith R., Goh H. S., Font A., Rosell R., Sun X. F., Zhang H., Benhattar J., Losi L., Lee J. Q., Wang S. T., Clarke P. A., Bell S., Quirke P., Bubb V. J., Piris J., Cruickshank N. R., Morton D., Fox J. C., Al Mulla F., Lees N., Hall C. N., Snary D., Wilkinson K., Dillon D., Costa J., Pricolo V. E., Finkelstein S. D., Thebo J. S., Senagore A. J., Halter S. A., Wadler S., Malik S., Krtolica K., Urosevic N. Kirsten ras mutations in patients with colorectal cancer: the “RASCAL II” study.
Br. J. Cancer
,
85
:
692
-696,  
2001
.
9
Maltzman T., Knoll K., Martinez M. E., Byers T., Stevens B. R., Marshall J. R., Reid M. E., Einspahr J., Hart N., Bhattacharyya A. K., Kramer C. B., Sampliner R., Alberts D. S., Ahnen D. J. Ki-ras proto-oncogene mutations in sporadic colorectal adenomas: relationship to histologic and clinical characteristics.
Gastroenterology
,
121
:
302
-309,  
2001
.
10
Rashid A., Houlihan P. S., Booker S., Petersen G. M., Giardiello F. M., Hamilton S. R. Phenotypic and molecular characteristics of hyperplastic polyposis.
Gastroenterology
,
119
:
323
-332,  
2000
.
11
Otori K., Oda Y., Sugiyama K., Hasebe T., Mukai K., Fujii T., Tajiri H., Yoshida S., Fukushima S., Esumi H. High frequency of K-ras mutations in human colorectal hyperplastic polyps.
Gut
,
40
:
660
-663,  
1997
.
12
Zhang B. H., Guan K. L. Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601.
EMBO J.
,
19
:
5429
-5439,  
2000
.